This is an R21 exploratory/developmental grant application to develop methods to quantify the content of alcohol metabolizing enzymes and variants (initially alcohol dehydrogenase) in human tissues (liver and esophagus). We believe that the application fits the R21 guidelines because we will use newly developed mass spectrometry proteomics methods to address the specific aims. The application is also in response to PA-05-074, Mechanisms of Alcohol-Induced Tissue Injury, because it provides important protein expression information to support the investigation of alcohol metabolizing enzyme genotypes that confer susceptibility or resistance to ethanol-induced cell/tissue damage. Our central hypothesis is that the expression/content of alcohol dehydrogenases (ADHs) and variants determine the pharmacokinetics and cellular toxicity of ethanol and acetaldehyde in tissues. In Aim #1, we will develop proteomics methods to quantify the expression of six ADHs in liver and esophagus tissue by triple quadruple linear ion trap LC/MS/MS and multiple reaction monitoring (MRM) with appropriate isotopic labeled ADH protein standards. We will develop a novel method of expressing isotopic labeled ADH protein standards in E. coli and using their peptide MRM transitions to quantify specific isoenzymes as well as groups of isoenzymes. In Aim #2, we have collected 22 frozen liver and 16 frozen esophagus samples (8 matched tumor and normal esophagus samples) from donors. The samples will be genotyped at the ADH loci. In Aim #3, we will perform a preliminary analysis to see if there is any correlation between ADH expression (Aim #1) and genotype (Aim #2) in tissues. We will estimate the individual contributions of six ADHs to alcohol metabolic capacity in liver and see if this capacity varies with genotype. We will assess the feasibility of extending the proteomics and genotyping analyses to other alcohol metabolizing enzymes like aldehyde dehydrogenase and cytochrome P450s involved in alcohol/acetaldehyde metabolism in humans.